Calpain I remains intact and intracellular during platelet activation. Immunochemical measurements with monoclonal and polyclonal antibodies.
Calpain I remains intact and intracellular during platelet activation. Immunochemical measurements with monoclonal and polyclonal antibodies.
复制标题
钙蛋白酶 I 在血小板活化过程中保持完整并保留在细胞内。
作者：
J. Samis;G. Zboril;J. Elce
作为了解血小板中钙蛋白酶（Ca2+激活的中性蛋白酶，EC 3.4.22.17）生理功能的一步，并且考虑到钙蛋白酶在血小板活化过程中转移到血小板外表面的一些建议，用免疫化学方法在静息细胞和凝血酶激活细胞中研究了该酶。 (1)制备了小鼠IgG1单克隆抗体，该抗体仅与人钙蛋白酶I的变性大亚基强结合，而与人钙蛋白酶II的大亚基结合弱。针对大鼠钙蛋白酶 II 产生的多克隆抗体是可用的，它除了与大鼠钙蛋白酶 II 强烈结合外，还与人钙蛋白酶 I 和 II 的大亚基大致相同地结合。 (2)使用这些抗体，发现只有在用0.1%皂苷透化后才能在悬浮的固定血小板中检测到钙蛋白酶，而在静息或活化的血小板的外表面或这些血小板的上清液中不能检测到钙蛋白酶。结论是钙蛋白酶在血小板活化过程中没有显着外化。 (3) 免疫印迹显示，仅当搅拌凝血酶激活的血小板以允许聚集时，才会发生较大的钙蛋白酶 I 亚基从 80 kDa 转化为 76-78 kDa，而在未搅拌的凝血酶激活过程中不会发生。虽然不能排除 80 kDa 形式的钙蛋白酶对可能的血小板底物（例如细胞骨架蛋白）的作用，但在聚集开始之前，钙蛋白酶肯定不会以 76-78 kDa 形式存在，这被认为是其活性形式。
As a step towards understanding the physiological function of calpain (Ca2+-activated neutral proteinase, EC 3.4.22.17) in blood platelets, and in view of some suggestions that calpain is transferred to the platelet external surface during platelet activation, the enzyme was studied with immunochemical methods in resting and thrombin-activated cells. (1) A mouse IgG1 monoclonal antibody was prepared which binds strongly only to the denatured large subunit of human calpain I, and weakly to that of human calpain II. A polyclonal antibody raised against rat calpain II was available which, apart from binding strongly to rat calpain II, binds to the large subunits of human calpain I and II about equally. (2) With these antibodies, it was found that calpain could be detected in fixed platelets in suspension only after permeabilization with 0.1% saponin, and could not be detected on the exterior surface of resting or of activated platelets, or in the supernatant media of these platelets. It was concluded that calpain is not significantly externalized during platelet activation. (3) Immunoblotting showed that conversion of the larger calpain I subunit from 80 kDa into 76-78 kDa occurred only when thrombin-activated platelets were stirred to permit aggregation, and did not occur during unstirred thrombin activation. Although an action of calpain in the 80 kDa form on possible platelet substrates such as cytoskeletal proteins cannot be excluded, calpain is certainly not present as the 76-78 kDa form, which is assumed to be its active form, until aggregation is initiated.